Polymorphism of prion protein gene (PRNP) in Nigerian sheep

ABSTRACT Polymorphism of the prion protein gene (PRNP) gene determines an animal’s susceptibility to scrapie. Three polymorphisms at codons 136, 154, and 171 have been linked to classical scrapie susceptibility, although many variants of PRNP have been reported. However, no study has investigated scrapie susceptibility in Nigerian sheep from the drier agro-climate zones. In this study, we aimed to identify PRNP polymorphism in nucleotide sequences of 126 Nigerian sheep by comparing them with public available studies on scrapie-affected sheep. Further, we deployed Polyphen-2, PROVEAN, and AMYCO analyses to determine the structure changes produced by the non-synonymous SNPs. Nineteen (19) SNPs were found in Nigerian sheep with 14 being non-synonymous. Interestingly, one novel SNP (T718C) was identified. There was a significant difference (P < 0.05) in the allele frequencies of PRNP codon 154 between sheep in Italy and Nigeria. Based on the prediction by Polyphen-2, R154H was probably damaging while H171Q was benign. Contrarily, all SNPs were neutral via PROVEAN analysis while two haplotypes (HYKK and HDKK) had similar amyloid propensity of PRNP with resistance haplotype in Nigerian sheep. Our study provides valuable information that could be possibly adopted in programs targeted at breeding for scrapie resistance in sheep from tropical regions.

The susceptibility of animals to scrapie is predominantly controlled by polymorphism in the PRNP gene [17]. Based on this, different single nucleotide mutations have been identified at codons 136 (A > V), 154 (R > H), and 171 (R > Q/H) of PRNP gene [18,19]. Amino acids at codons 141 and 154 have been found to be associated with various forms of classical scrapie through modification of the configuration of prion protein [20,21]. Changes at codon 136 from A to V has been reported to increase susceptibility to scrapie while variations at codon 171 from Q to R cause resistance in sheep [18,22].
It was believed that the ancestral PRNP gene was ARQ/ARQ (ARQ/ARQ wild type) in sheep [23]. There are six primary forms of the wild-type allele which include ARQ, VRQ, AHQ, ARH, ARK, and ARR [24]. Also, the genotypes associated with the admixture of these alleles are classified into five groups based on their degrees of resistance to scrapie [25]. Recent studies have shown that amino acid substitution in some nucleotide positions were associated with resistance to scrapie and TSE in ARQ/ARQ sheep [26].
Nigerian sheep are reared in the drier agro-climatic zones of the country with an estimated population of 27 million [49]. There are four major breeds of Nigerian sheep: Yankasa, Uda, Balami, and West Africa Dwarf [50]. Sheep have socio-economic values in Nigeria as they form part of the livelihoods of small ruminant farmers that are majorly rural dwellers [51]. Environmental factors of Nigeria impose health-related risks on the lives of human and animals which require urgent attention [52].
Several studies have been carried out on Nigerian sheep using genetic techniques such as microsatellite DNA polymorphism [53][54][55][56], polymorphism study on Myostatin (MSTN) [57], and transferrin and haemoglobin [58]. The survey conducted on the occurrence of scrapie in Jos within the central part of Nigeria [59] is not sufficient to conclude on the absence of scrapie in Nigerian sheep. There is no report on polymorphism of the PRNP gene in Nigerian sheep, despite its significant effects on scrapie, the economic importance of Nigerian sheep, and transmission of the diseases from animals to humans.
Herein, we examined the genotype and allele frequencies of PRNP polymorphisms in 126 Nigerian sheep and compared the sampled population with previous studies on scrapie-affected animals in different breeds of sheep. Subsequently, we evaluated the linkage disequilibrium (LD) and analysed haplotypes of the PRNP polymorphisms in Nigerian sheep. Finally, we computed the biological impact, which includes the protein structure and functions of nonsynonymous SNPs via PolyPhen-2, PROVEAN, and AMYCO investigations.

Blood sample collection and DNA extraction
We collected 10 ml of blood samples from 126 sheep (61 females and 65 males) from four different states in Nigeria namely, Kaduna State (n = 18 female; n = 15 male), Katsina State (n = 7 female; n = 8 male), Sokoto State (n = 9 female; n = 15 male), and Taraba State (n = 26 female; n = 28 male) (Supplementary Fig S1). During sample collection, we excluded sheep with close relationships after obtaining information from the herders. The whole blood samples were stored at −20 •C prior to DNA extraction. Genomic DNA was extracted at Kunming Institute of Zoology, Chinese Academy of Sciences (CAS) using the phenolchloroform method [60]. This was followed by the quantification using the Thermo Scientific™ NanoDrop 2000 spectrophotometer to evaluate the purity of the obtained DNA. Further, the quality of the total genomic DNA was checked by running gel electrophoresis using a 2% agarose gel with a 2 Kilobase (kb) DNA ladder marker. The 126 Nigerian sheep samples were then sequenced using the Sanger method.
The 25 µl PCR mixture and sequencing reactions contained 1 μl of genomic DNA, 10 pmol of each primer, 2.5 mM dNTPs, and 5 units of Takara Taq DNA polymerase in a 10 pmol reaction buffer containing 1.5 mM MgCl2.
The PCR condition was as follows: 96°C for 5 min, 35 cycles of denaturation at 96°C for 30 s, 57°C for 15 s, 72°C for 1 min 30 s, and final extension of 72°C at 4 min. PCR products were purified for sequencing analysis with a QIAquick Gel Extraction Kit (Qiagen, Valencia California, USA). The PCR products were bidirectionally sequenced in an ABI 3730×L sequencer (Applied Biosystems, Foster City, California, USA).

Statistical analysis
Hardy Weinberg Equilibrium (HWE), Linkage Disequilibrium (LD) and haplotype distributions of the PRNP gene in Nigeria sheep were performed using DNA SNP Version 6.12.03 [61]. The genotype differences, allele, and haplotype frequencies of the PRNP gene were analysed by chi-square test (χ2) or Fisher's exact test using SPSS v21.0.

Identification of polymorphic sites of the PRNP gene in 126 Nigerian sheep
To achieve this, the open reading frame (ORF) of the PRNP gene in 126 Nigerian sheep was sequenced. The ORF contained 771 bp in length and occupied similar position with the PRNP gene of Ovis aries retrieved from the NCBI database (Gene ID:EF153678). In this study, we identified 19 SNPs, including 14 nonsynonymous SNPs being the T718C a novel SNP. (Supplementary Table S1 and Supplementary Figure  S2). Table 1 shows the genotype and allele frequencies of the 13 non-synonymous SNPs of PRNP in Nigerian sheep. Interestingly, all genotype frequencies of the identified SNPs conform to Hardy-Weinberg Equilibrium (HWE). Also, we examined the LD among the 19 SNPs detected with others from previous studies using Lewontin's D' (|D'|) values ( Table 2). The majority of the SNPs showed negative LD ranging from −0.018 to −0.188, except c.711 G > C which showed strong positive LD values of 0.135 and 0.095 with some SNPs.
Haplotype frequency of the 14 PRNP nonsynonymous SNPs were further examined as shown in Table 3. Based on the haplotype analysis, we found eight (8) major haplotypes with the haplotype QMAGGHRRQQYNNS having the highest frequency (58.0%) followed by QMAGGRRRQQYNNS (13.7%) and QMAGGHRRQQDKKS (1.5%).

Estimation of potential scrapie vulnerability in Nigerian sheep
To estimate potential scrapie vulnerability in Nigerian sheep, we compared the genetic distribution of scrapierelated SNPs (R154H and S240P) between Nigerian sheep and scrapie-affected sheep in other countries. Previous studies related to SNPs of ovine PRNP gene were selected to estimate the susceptibility in Nigerian sheep [30,40].
There was a significant difference (P<0.05) in allele frequencies of scrapie-affected Italian sheep and healthy Nigerian sheep counterpart at PRNP codon 143 ( Figure 1a). However, no significant (P = 0.0064) difference was recorded between the allele frequencies of scrapie-affected Chinese sheep and Nigerian sheep at codon 143 ( Figure 1a).
Moreover, the allele frequencies of scrapie-affected Chinese and Nigerian sheep had no significant (P = 0.152) difference at codon 154 ( Figure 1b).

Comparison of haplotype and genotype frequencies at PRNP codons 143, 154 and 171 between scrapie-affected and healthy sheep
The ovine PRNP haplotypes of codons 143,154 and 171 in Nigerian sheep were used for comparison with other previously published works on PRNP in sheep. Moreover, the PRNP haplotypes were compared with those previously reported in two countries i.e. Italian and Spanish sheep [7,40]. In the considered countries, the ARR and AHQ haplotypes were significantly different between the scrapie-affected and healthy sheep (Table 4). In Italian and Spanish sheep, ARQ haplotype was present but was not in Nigerian sheep. The genotypes ARR/ARR and ARQ/ARQ were significantly different between the scrapie-affected and healthy sheep.

Assessment of nonsynonymous SNPs of the PRNP gene in Nigerian sheep
PolyPhen-2 is an online tool used to predict the outcome of an amino acid substitution caused by nonsynonymous SNPs on the structure and function of proteins [64]. Based on our polymorphism results, the effects of 14 non-synonymous SNPs identified were assessed via PolyPhen-2. Three different predictions were observed for the 14 non-synonymous SNPs which include benign: T112M (0.000), S127G (0.000), S129G (0.000), H143R (0.241), H171Q (0.000), N176K (0.002), probably damaging: A116P (1.000), R151G (1.000), R154H (0.998), Y172D (1.000), and possibly damaging: Q101R (0.621), S240P (0.827) as shown in Table 5. We predicted the biological impact of the nonsynonymous SNPs identified with PROVEAN [65]. Interestingly, the nonsynonymous SNPs of the PRNP gene identified in this study were predicted as 'neutral' (Table 5). Finally, we explored the amyloid propensity of ovine prion protein using the alleles of nonsynonymous SNPs. Based on this, we analysed the prion protein of these alleles and the HYNN haplotype was estimated with 0.00 values by AMYCO. Additionally, we evaluated the prion protein of Nigerian sheep. We classified amino acid sequences of PRNP in Nigerian sheep into three haplotypes (HYKK, HDKK and RYNN) considering the alleles of the 14 nonsynonymous SNPs. Based on AMYCO score, Haplotypes HYKK and HDKK when evaluated had a value of 0.00, while RYNN haplotype gave a value of 0.08 (Figure 2).

Discussion
Scrapie is a fatal disease in sheep but the distribution of the PrP genotypes could serve as a means to prevent subsequent reoccurrence in breeding programme. This study analysed the nucleotide sequence of the PRNP gene in 126 Nigerian sheep and revealed 19 SNPs of which 14 were non-synonymous. Interestingly, one novel SNP (T718C) was identified.
The    related to vulnerability or resistance to scrapie in sheep [66][67][68]. The allelic variants (VRQ and ARQ) of PRNP at codons 136, 154 and 171 have been reported to correlate with high resistance to scrapie [69,70]. Moreover, the ARR allele was associated with low resistance to scrapie [71]. In contrast, the VRQ allele at the same codon had low survivability after exposure to scrapie [72]. Based on this, there is a need to develop breeding programs that will increase the frequency of the ARR allele [73,74]. It is believed that increasing such scrapie-resistant genotypes will enhance scrapie control.
In this present study, the non-synonymous substitutions i.e. Q101R, T112M, A116P, S127G, S129G, H143R, R151G, R154H, H171Q, Y172D, N176K, and S240P were identified ( Table 5). The mutation at codon 127 was highly polymorphic similar to the trend detected in scrapie susceptibility in three native Ethiopian sheep breeds [46]. It has been reported that amino acids at codons 126 and 127 are found in highly conserved glycine-rich motif.
GAVVG126G127LGGYMLG could reduce development of prion disease via blockage of amyloid fibril formation [13]. In a previous, polymorphism at codon 127 was Table 3. Haplotype frequencies of fourteen (14) non-synonymous single nucleotide polymorphism of PRNP gene in Nigerian sheep.
Haplotypes A302G C335T G346C A379G A385G A428G C451G G461A A512G T513G T514G G526A C528A T718C N = 262 Q101R T112M A116P S127G S129G H143R R151G R154H H171Q H171Q Y172D N176K N176K S240P found to play a crucial role in the normal cellular function of PRNP [75]. Moreover, the genotypes at codon 171Q/K are also related to scrapie susceptibility [76]. Furthermore, PolyPhen-2 and PROVEAN were used to predict the impacts of 14 non-synonymous SNPs identified in this study. Using PolyPhen-2, three different predictions were observed for the 14 nonsynonymous SNPs which include benign: T112M (0.000), S127G (0.000), S129G (0.   populations were '(neutral or non-deleterious)' using PROVEAN. The discrepancies in the prediction outcomes of the two software might be due to variations in algorithms that reproduce the effect on the position of the protein [64,77] and difference in their mode of analyses [78]. We investigated the amyloid propensity of sheep prion protein based on the alleles of nonsynonymous SNPs of the PRNP gene in sheep. We found out that the amyloid formation of PRNP in two of the haplotypes (HYKK and HDKK) was similar to the resistance haplotype (HYNN) to prion diseases in this study. However, one of the haplotypes (RYNN) had a higher degree of amyloid formation value than the resistance haplotype ( Figure 2).

Conclusion
In conclusion, the polymorphism identified in this study show that sheep in Nigeria are susceptible to scrapie because of the variations detected at codons 154 and 171. The information obtained about the variation in allelic frequencies of PRNP gene in Nigerian sheep could assist into designing valuable scrapieresistance breeding projects especially for sheep in a tropical region like Nigeria, thereby reducing disease outbreaks caused by scrapie and the subsequent increase in cost of production.

Acknowledgments
We appreciate the important contributions of the Central Abattoir, Ibadan and the Ministry of Agriculture and Rural Development, Oyo State, in Nigeria, who made possible the collection of sheep samples for this study. We recognize all of those who assisted in the success of this study.

Disclosure statement
No potential conflict of interest was reported by the authors.

Informed consent statement:
Not applicable.